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Engineered microfluidic chips combined with human pluripotent stem cell (hPSC)\u2010derived neurons provide valuable tools for targeted in vitro research on axons to improve understanding of disease mechanisms and enhance drug development. Here, a polydimethylsiloxane (PDMS) microfluidic chip integrated with a light patterned substrate is utilized to achieve both isolated and unidirectional axonal growth of hPSC\u2010derived neurons. The isolation of axons from somas and dendrites and robust axonal outgrowth to adjacent, axonal compartment, is achieved by optimized cross\u2010sectional area and length of PDMS microtunnels in the microfluidic device. In the axonal compartment, the photoinscribed nanotopography on a thin film of azobenzene\u2010containing molecular glass efficiently guides the growth of axons. 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